Acute lymphoblastic leukemia (ALL) is the most common form of childhood malignancy. Champlin et al., Blood, 73, 2051 (1989). Each year about 1250 children less than 15 years of age are found to have acute lymphoblastic leukemia. Champlin et al., cited supra. Recently, dramatic improvements in the multiagent chemotherapy of children with ALL have resulted in cure rates of 70-75%. Poplack et al., Pediatric Clinics of North America, 35, 903 (1988). However, despite these recent improvements, as many as 1 in 5 patients will eventually suffer leukemic relapse. Riehm et al., Haematol. Blood Transf., 33, 439 (1990). This occurrence of relapsed patients equates to 250 cases/year and is equivalent to the number of newly diagnosed cases of childhood acute nonlymphoblastic leukemia, medulloblastoma, and rhabxomyosarcoma Furthermore, this relapse rate surpasses the number of newly diagnosed cases of childhood Ewings sarcoma, osteogenic sarcoma, hepatoma, and germ cell tumors. The unsatisfactory outcome of this population makes a significant contribution to overall pediatric cancer mortality, despite the excellent outcome for the substantial majority of children with ALL.
Currently, the major challenge in the treatment of childhood ALL is to cure patients who have relapsed despite intensive multiagent chemotherapy. Champlin et al., cited supra. For patients who have relapsed while on therapy or shortly after elective cessation of therapy, the overall survival is very poor. Poplack et al., cited supra. Treatment of these relapsed children has generally employed either intensive chemotherapy to achieve a second remission, subsequent use of either nonablative chemotherapy or ablative radiochemotherapy and bone marrow transplantation (BMT). Kersey et al., N Engl. J. Med., 117, 461 (1987). However, recurrence of leukemia is the major obstacle to the success of either approach. Dicke et al., Clin. Hematol., 15, 86 (1986).
Furthermore, treatment of these relapsed patients by the intensification of cytotoxic therapy using conventional drugs-will likely cause overlapping toxicities and may result in delays which may erode the intensity of therapy. Consequently, the development of new potent anti-ALL drugs and the design of combinative treatment protocols utilizing these new agents, have emerged as focal points for research in the therapy of relapsed ALL.
Acute myeloid leukemia (AML) is the most common form of acute leukemia in adults and the second most frequent leukemia in children, accounting for 20-25% of acute childhood leukemias. Priesier et al., Blood, 80, 2600 (1992). Though the majority of patients with myeloid leukemias initially respond to intensive chemotherapy regimens, most will relapse and eventually succumb to their disease. Additionally, attempts to identify useful and specific prognostic factors to effectively stratify good and poor outcome AML patients have generally not been successful, with the result that all patients receive very intensive therapy at the price of great morbidity. Furthermore, contemporary multiagent chemotherapy regimens for AML fail to cure more than half of the patients because of multidrug resistance of leukemia cells and often lead to potentially fatal systemic toxicity. Gale et al., Sem. Hematol., 24, 40 (1987).
Finally, although allogeneic bone marrow transplantation has been demonstrated to be an effective therapy for many patients with myeloid leukemia, its application is limited by the availability of suitable HLA-matched and MLC-unreactive donors. Woods et al., J. Clin. Oncol., 11, 1448 (1993). In autologous bone marrow transplantation for childhood AML, gene marker studies have indicated that subclinical disease in unpurged xe2x80x9cremissionxe2x80x9d marrow harvested for transplantation contributes significantly to disease recurrence. Brenner et al., Lancet, 341, 85 (1993). Myeloablative chemotherapy or supralethal radiochemotherapy followed by allogeneic or autologous bone marrow transplantation are associated with considerable morbidity and mortality and fail to substantially improve the overall survival of AML patents, underscoring the need for rational, drug design-based therapies for AML. Yeaper et al., New Engl. J. Med., 315, 141 (1986); Woods et al., cited supra.
Another disease of the immune system is acquired immunodeficiency syndrome (AIDS). Infection with the human immunodeficiency virus type I (HIV-1) constitutes a worldwide public health problem. Venkatesan, Science, 241, 1481 (1988). The critical basis for the immunopathogenesis of HIV infection is the depletion of the CD4+ helper/inducer subset of T-cells, resulting in profound immunosuppression. See Dahlgleish et al, Nature, 312, 763 (1984); Fauci, Clin. Res., 32, 491 (1985); Ho et al., N. Engl. J. Med., 317, 278 (1987). HIV has a selective tropism for CD4+ T-cells and macrophages which is mediated by interaction of its envelope (env) protein gp 120 with an essential component of the cell surface receptor for HIV-1, the CD4 antigen. Lasky et al., Science, 233 209 (1986). After HIV binds to the first domain of the CD4 molecule via the external envelope glycoprotein gp120, the virus is internalized and uncoated. Fauci, Science, 239, 617(1988). Once uncoated, the viral genomic RNA is transcribed to DNA by the enzyme reverse transcriptase. The proviral DNA is then integrated into the host chromosomal DNA. After integration of the provirus, the infection may assume a latent phase or the proviral DNA may transcribe viral genomic RNA and messenger RNA. Protein synthesis, processing, and virus assembly occur with budding of the mature virion from the cell surface.
At present, AIDS is incurable and treatment modalities that reduce HIV-1 replication in vivo by using reverse transcriptase inhibitors such as zidovudine/ZDV (formerly termed azidothymidine/AZT) and dideoxyinosine (ddI) cause substantial side effects. Yarchoan et al., Blood, 78, 859 (1991). Although ZDV delays the disease progression in HIV-1 seropositive asymptomatic individuals and has improved the survival of patients with AIDS and AIDS-related complex (ARC), the therapeutic response is frequently transient. Volberding et al., N. Engl. J. Med., 322, 941 (1990); Fischl et al., Ann. Intern. Med., 112, 727 (1990); Fischl et al., N. Engl. J. Med., 317, 185 (1987). Moreover, variants of HIV-1 that are resistant to ZDV emerge to thwart the success of continued therapy. Erice et al., Clinical Infectious Disease, 18, 149 (1994). Recent data indicate that resistance among HIV-1 isolates also emerges during dideoxyinosine (ddI) therapy. St. Clair et al., Science, 253, 1557 (1991). These characteristics confirm the resilience of HIV-1 and the need for more powerful strategies against this virus.
Drug targeting is a potentially attractive new approach to killing malignant or HIV-infected cells, an approach which can leave normal or uninfected tissue or cells unharmed. A decisive breakthrough in drug targeting was the advent of hybridoma technology, making many monoclonal antibodies (MoAbs) available in essentially limitless supply. To construct therapeutic reagents with selectivity for certain populations of cells, MoAbs or other cell targeting proteins are linked to bioactive moieties to form biotherapeutic agents referred to as immunoconjugates, immunotoxins or fusion proteins, which can combine the selectivity of the targeting moiety with the potency of the bioactive moiety. The choice of MoAb (or other targeting moiety) is based on the surface antigen profile of a target cell.
For the past decade, these types of biotherapeutic agents have been under investigation for the treatment of various cancers. Although these biotherapeutic agents have shown some potential to provide safe and effective therapy for human disease, many difficulties remain. Ideally, consistently locatable and reliable markers on target cells would permit the binding portion of biotherapeutic agents to completely avoid non-target tissue. In reality, cross-reactivity with antigens expressed by vital life-maintaining organs often gives rise to unacceptable complications in in vivo applications. There is also the potential that patients will demonstrate immune responses to the separate components of the biotherapeutic agents even though they may already be immunosuppressed by the course of their disease. Moreover, the cytotoxicity obtained in in vitro studies may be limited in clinical application due to a lack of potency in doses that can be tolerated by the patient Finally, solid tumors are difficult to penetrate thoroughly, and in hematologic malignancies, residual disease can cause relapse despite easier access to target cells in leukemias and lymphomas.
Toxicity studies using immunotoxins in mice and monkeys have not been predictive of the toxicity of the immunotoxins in clinical trials. For example, while no neurotoxicity was observed in monkeys treated with ricin A chain immunotoxins directed to B-cell surface antigens CD19 or CD22, when these immunotoxins were used in patients with lymphoma, a significant fraction showed peripheral neuropathy as well as aphasia (loss of speech). Similarly, no neurotoxicity was observed in preclinical animal studies using a recombinant ricin A chain immunotoxin of 454A12 mouse antitransferrin receptor monoclonal antibody or a natural pseudomonas exotoxin immunotoxin of OVB3 mouse anti-adrenocarcinoma monoclonal antibody. However, both immunotoxins caused lethal neurotoxicity with severe encephalopathy and brainstem inflammation when used in patients with cancer. Grossbard et al., Blood, 80, 863 (1992); Hertler et al., J. Clin. Oncol., 7, 1932 (1989).
PAP has been used as the ribosomal-inhibitory (cytotoxic) moiety of an anti-CD19 immunotoxin in Phase I/II clinical trials of adult and pediatric patients with acute lymphoblastic leukemia under an Investigational New Drug Application (BB-IND-3864) approved by the Food and Drug Administration. Uckun F. M., Brit. J. Haematol., 85, 435 (1993). Anti-CD19 PAP has been developed as an anti-leukemia agent since 1984 and generated very promising results in preclinical leukemia models, which provided the basis for ongoing clinical investigations. Uckun et al., Leukemia, 7, 341 (1993); Uchcm et al., Journal of Exp. Med., 163, 347 (1986).
In a recently completed Phase I/II study, 18 patients with leukemia received escalating doses of anti-CD19 PAP at dose levels ranging from 0.1 xcexcg/kg/day to 250 xcexcg/kg/dayxc3x975 days and 10 patients received anti-CD19-PAP at a fixed dose level of 100 xcexcg/kg/dayxc3x975 days. Uckun F. M., Brit. J. Haematol., 85, 435 (1993). A maximum tolerated dose was not reached at the highest dose level of 250 xcexcg/kg/dayxc3x975 days. Patients were given 1 hour i.v. infusions of anti-CD19-PAP on each of five days during one to three courses of treatment. Toxicities included capillary leak syndrome and myalgias. Importantly, no significant hepatic, renal, cardiac, or neurologic toxicity has been observed, and patients have not developed an immune response to either the PAP or monoclonal antibody moiety of anti-CD19 PAP. Thus, the clinical toxicity profile of PAP administered as an immunoconjugate is very different from the reported toxicity profiles of other RIPs. Of the 24 evaluable patients, 5 achieved a complete remission, 2 achieved a partial remission, 5 had partial responses but did not achieve remission, 9 had stable disease and only 3 progressed while on therapy. Four patients received treatment for minimal leukemia burden: therefore they are not evaluable for objective response. Thus, anti-CD19 PAP was able to penetrate bone marrow, liver, spleen, and lymph nodes leading to selective eradication of CD19-positive leukemia cells.
It has been reported that HIV-1 replication in normal CD4+ T cells can be inhibited in vitro by PAP. Zarling et al., Nature, 347, 92 (1990). Notably, targeting PAP to CD4+ T cells in vitro by conjugating it with MoAbs reactive with CD4+ T cells increased its potency  greater than 1,000-fold in inhibition of HIV-1 replication. Zarling et al., supra. Subsequent studies using clinical isolates of AZT-sensitive and AZT-resistant HIV-1 demonstrated that G17.2(anti-CD4)-PAP immunoconjugate exhibits potent anti-HIV activity against all isolates at nanomolar concentrations (Erice et al., Antimicrobial Agents and Chemo., 37: 835 (1993)). However, the stability and efficacy of the G17.2(anti-CD4)PAP immunoconjugate in vivo is unclear.
Therefore, a need exists for an anti-T cell PAP immunotoxin with improved stability that is efficacious in vivo. Moreover, there is a continuing need for immunotoxins and methods of their use to target and inhibit or eliminate cell populations associated with various T cell-specific pathologies.
The present invention provides a biotherapeutic agent, e.g., an immunoconjugate or immunotoxin, comprising a monoclonal antibody specific to mammalian, e.g., human, T-cell/myeloid antigen CD7, linked to an effective amount of moiety, e.g., a polypeptide or a toxin, which has biological activity. These agents are active both in vitro and in vivo, and are useful to treat CD7+ T cell-specific diseases, such as certain cancers and certain viral infections, e.g., HIV infections associated with AIDS or ARC. As used herein, the term monoclonal antibody (MoAb) includes a fragment, a subunit or a derivative thereof, which is preferably covalently bonded or cross-linked to a biologically active moiety. Preferably, the moiety is pokeweed antiviral protein (PAP). The term xe2x80x9cpokeweed antiviral proteinxe2x80x9d includes any moiety, e.g., a pokeweed protein, subunit, variant or derivative thereof such as PAP-II, PAP-S, and recombinant PAP, that has at least about 1%, preferably about 10%, and more preferably about 50%, the activity of native, purified pokeweed antiviral protein. The activity of a preparation of pokeweed antiviral protein can be determined by methods well known to the art, including methods described hereinbelow.
Thus, to treat cancer, the immunotoxin of the invention preferably comprises a cytotoxic amount of pokeweed antiviral protein. To inhibit or treat viral infections, the immunoconjugate of the invention preferably comprises an amount of pokeweed antiviral protein that is effective to inhibit viral infection and/or replication.
It is preferred that the immunoconjugate or immunotoxin of the present invention employs the monoclonal antibody TXU-7 or a biologically active subunit, fragment or derivative thereof, which binds to the CD7 antigen present at the surface of mammalian T-cell/myeloid cells, for example, the CD7 antigen present on the surface of leukemic blasts from T-cell ALL, AML and T-lineage lymphoma patients. A xe2x80x9cbiologically activexe2x80x9d subunit or fragment of a monoclonal antibody has at least about 1%, preferably at least about 10%, and more preferably at least about 50%, of the binding activity of the monoclonal antibody. More preferably, the antibody utilized in the practice of the present invention has the binding specificity of the monoclonal antibody produced by hybrid cell line ATCC HB-12260.
Unlike immunoconjugates that rely on the expression of HIV-1 envelope proteins on infected cells to provide them with binding targets, the immunoconjugate of the present invention targets pokeweed antiviral protein to uninfected or latently infected CD7+ cells using monoclonal antibodies against normal antigens on CD7+ cells. It had been previously discovered by Applicant, and described in U.S. patent application Ser. No. 07/979,470, which application is incorporated herein by reference, that the internalization of protein antiviral protein-monoclonal antibody conjugates by monoclonal antibody receptor-mediated endocytosis results in increased delivery of pokeweed antiviral protein through the plasma membrane, as compared to the non-specific uptake that occurs at high pokeweed antiviral protein concentrations.
However, the pokeweed antiviral protein immunoconjugates disclosed in the ""470 application display very poor in vivo stability and showed no anti-HIV activity in SCID mouse models of human AIDS. In contrast, as described hereinbelow, the immunoconjugate of the present invention showed potent anti-HIV-1 activity in a SCID mouse model of human AIDS without causing systemic toxicity. Moreover, in cynomolgus monkeys, the immunoconjugate of the present invention showed favorable pharmacokinetics with an elimination half-life of 8.1-8.7 hours. The monkeys treated with TXU-PAP at dose levels of 50 xcexcg/kg/dayxc3x975 days or 100 xcexcg/kg/dayxc3x975 days tolerated the therapy very well, without any significant clinical compromise or side effects, and at necropsy no gross or microscopic lesions were found. Thus, the immunoconjugate of the present invention exhibits surprising in vivo stability as measured by longer serum half-life and greater systemic exposure.
Hence, the present invention also provides a method to treat viral infection or inhibit viral replication in mammalian cells. The method comprises treating mammalian cells in vitro or a mammal having, or at risk of, a viral infection with an effective amount of the immunoconjugate of the present invention. One embodiment of the invention is a method to inhibit HIV replication or reduce viral burden in mammalian cells of the myeloid lineage and T-cells; thereby providing a method to treat patients with AIDS, ARC or asymptomatic patients infected with HIV-1 who have not yet developed AIDS. The immunoconjugate of the present invention may also be utilized in combination with at least one of the more conventional anti-AIDS agents, such as an anti-viral nucleoside analog, e.g., the reverse transcriptase inhibitor zidovudine (ZDV), without causing undesired side effects. The present method is especially suited for the treatment of patients infected with HIV stains that have become ZDV resistant.
Moreover, the present immunoconjugate may also provide the basis for an effective method to inhibit other lentiviruses (HTLV-1, etc.) and viruses other than lentiviruses that infect CD7+ cells, viruses including, but not limited to, members of the herpes virus group (HSV, CMV, EBV), influenza viruses, rhinoviruses, papovaviruses (e.g., human papilloma), adenoviruses, hepatitis virus, and the like.
The invention further provides an immunotoxin useful to treat diseases or pathologies associated with undesirable T-cell proliferation, either alone or in combination with conventional therapies for such afflictions. Such pathologies include cancers, such as T-cell leukemias or lymphomas, acute myeloid leukemia, organ rejection, rejection of bone marrow transplants or autoimmune diseases such as systemic lupus erythematosus, rheumatoid arthritis, non-glomerular nephrosis, psoriasis, chronic active hepatitis, ulcerative colitis, Crohn""s disease, Behret""s disease, chronic glomerulonephritis (membranous), chronic thrombocytopenic purpura, and autoimmune hemolytic anemia The immunotoxin comprises a monoclonal antibody specific for the human T-cell myeloid antigen CD7, or a biologically active fragment or subunit thereof, linked to a cytotoxic amount of pokeweed antiviral protein. Preferably, the immunotoxin of the present invention employs the monoclonal antibody TXU-7 or a biologically active fragment or subunit thereof, which binds to the CD7 antigen present at the surface of mammalian cells.
Yet another embodiment of the present invention is a therapeutic method for the treatment of cancer. The method comprises parenteally administering to a mammal who is so afflicted with an amount of a pharmaceutical composition comprising an immunotoxin comprising monoclonal antibody TXU-7, or a biologically active fragment or subunit thereof, covalently linked to pokeweed antiviral protein, in combination with a pharmaceutically acceptable carrier. The amount of the composition administered is effective to inhibit or treat the cancer, e.g., it is a cytotoxic or an anti-neoplastic amount Preferably, the cancer to be treated is T-cell leukemia, lymphoma or acute myeloid leukemia (AML). The term xe2x80x9ccytotoxic amountxe2x80x9d is defined to mean an amount of pokeweed antiviral protein that is toxic to the target cell once the immunotoxin has associated with the cell.
Peripheral cancer cells that lack the target antigen may present complications in the treatment of certain patients. In these cases, combined or adjunctive therapies that exploit the diverse cytotoxic mechanisms offered by conventional chemotherapy or radiation can assist in the elimination of any cancer cells that lack the target antigen as well as in the suppression of immunotoxin-resistant mutants. Thus, one embodiment of the present invention comprises the administration of TXU-7-pokeweed antiviral protein in conjunction with, e.g., before, during or after, or a combination thereof, the administration of an effective amount of one or more conventional antineoplastic agents. Preferably, the antineoplastic agent employed is an anti-metabolite or a class I or a class III immunosuppressive agent. Preferably, the antineoplastic agent employed is cytarabine, methotrexate, trimetrexate, 5-fluorouracil, mercaptopurine, thioguanine, 5-azacytidine, floxuridine or 2xe2x80x3-chlorodeoxyadenosine, cyclophosphamide or etoposide. More preferably, the antineoplastic agent employed is cyclophosphamnide or etoposide. It is also preferred that the antineoplastic agent be combined with a pharmaceutically acceptable liquid carrier at a concentration of from about 10 mg/ml to about 30 mg/ml. In this embodiment of the invention, it is preferred that the antineoplastic agent, e.g., cyclophosphamide or cytarabine, be administered intravenously. Preferably, cyclophosphamide is administered at the rate of 0.5-3.5 L/M2/24 hours.